The present invention relates to immunological products derived from molecular cloning, and to their method of use. More particularly, this invention relates to immunological polypeptides useful as diagnostic products and vaccines in the detection of and vaccination against viral etiological agents of acquired immunodeficiency syndrome. This invention also relates to polypeptides of AIDS retrovirus reverse transcriptase and to their method of use.
Analysis of the immune response to a variety of viral infectious agents has been limited by the fact that it has often proved difficult to culture such pathogens in quantities sufficient to permit the isolation of viral polypeptides which may be used (a) as a diagnostic product to detect an immune response to such pathogens or (b) as a vaccine to confer resistance to infection by such pathogens.
The advent of molecular cloning has overcome some of these limitations by providing a means whereby gene products from pathogenic agents can be expressed in virtually unlimited quantities in a non-pathogenic form.
Although antigens from the viruses for influenza (1), foot and mouth disease (2), hepatitis (3), vesicular stomatitis virus (4) and rabies (5) have been reported to be expressed in E. coli, several laboratories have reported that the surface antigen for hepatitis B expressed in prokaryotes is not immunologically reactive with antisera to the naturally occurring antigen (6).
Acquired immunodeficiency syndrome (hereinafter referred to as AIDS) is a devastating disease of the adult immune system which significantly affects cell-mediated immunity. The disease is manifested by a profound lymphopenia which appears to be the result of a loss of T-lymphocytes that have the helper/inducer phenotype T4 as defined by the monoclonal antibody OKT4 (7). Other clinical manifestations include opportunistic infections, predominantly Pneumocystis carinii pneumonia, and Karposi""s sarcoma (7).
Pre-AIDS, a syndrome that often precedes the onset of AIDS, is characterized by chronic generalized lymphadenopathy. It has been reported that about 10% of patients with pre-AIDS develop AIDS (8).
The predominant risk group for AIDS includes homosexual and bisexual males, intravenous drug abusers, recipients of blood components (primarily hemophiliacs receiving treatment with Factor VIII) and recipients of blood transfusions (7). The epidemiology of this syndrome indicates that AIDS is caused by an infectious agent which is transmitted by intimate contact or contact with blood or blood products (7).
A human retrovirus related to the previously described human T-lymphotropic viruses HTLV-I and HTLV-II, is the causative agent for AIDS (8, 9, 10). Several laboratories originally isolated and propagated retrovirus associated with AIDS patients (11) and propagated in a permissive T-cell line (12). Similarly, Luc Montagnier of the Pasteur Institut in Paris has found a virus in AIDS patients designated LAV (lymphadenopathy associated virus). Some authors have reported that LAV may be similar or identical to HTLV-III (13, 14). A virus isolated from an AIDS patient, designated ARV (AIDS Related Virus), was reported to have been identified and cloned (15). These and other AIDS retroviruses are hereinafter referred to as AIDS associated retrovirus.
HTLV-III is a retrovirus with a genome comprising approximately 10 kb of RNA. The virus particle contains a capsid consisting of a number of proteins. The primary core protein is designated p-24 and has a corresponding molecular weight of about 24,000 daltons. The p-24 core protein is synthesized in vivo as part of a precursor polypeptide encoded by the gag region of the HTLV-III genome. This precursor polypeptide is processed in the infected cell to form p-24 and other viral proteins. In addition, an envelope protein designated gp-41 (MW 41,000 daltons), is also a constituent of the virus particle and is encoded in the env region of the HTLV-III genome.
Antibodies to the envelope protein gp-41 of HTLV-III have been detected in serum from AIDS and pre-AIDS patients. Approximately 88% of AIDS and 79% of pre-AIDS patients have detectable antibodies to HTLV-III envelope protein (16). One author has reported that there is a 90-100% sero-conversion to such antibodies (8). In addition to antibodies to the gp-41 envelope protein, antibodies to the HTLV-III core protein p-24, and the HTLV-III proteins designated p-55, p-60 as well as the glycoproteins gp-65, gp-120 and gp-160 have been detected in patients with AIDS (16, 17). The detection of antibodies to these and other as yet unknown antigens is, therefore, a significant indication of exposure to or productive infection by the agent responsible for AIDS.
In addition, certain of these viral polypeptide sequences may be used as a vaccine to induce the production of neutralizing antibodies conferring resistance to AIDS infection. A need exists for variant sequences such as fusions of the viral polypeptide with highly immunogenic polypeptides, in order to facilitate induction of a high titer immune response, as well as for deletions of undesired viral sequences.
Accordingly, it is an object herein to express, in a prokaryotic host, viral antigens of an AIDS associated retrovirus which are non-pathogenic and which may be used as diagnostic product to detect AIDS.
It is a further object of the present invention to prokaryotically express a diagnostic product to detect AIDS consisting of variant composite polypeptides of naturally occurring AIDS related polypeptide sequences.
Further, an object of the present invention is to express in mammalian cell culture AIDS associated polypeptides or variants, including fusion polypeptides of an AIDS associated polypeptide which may be used as a vaccine against AIDS.
Still further, an object of the present invention is to express AIDS RNA dependent DNA polymerase (reverse transcriptase) for use in an assay to identify transcriptase inhibitors.
In accordance with this invention, DNA encoding AIDS-associated polypeptides is identified and employed in recombinant prokaryotic or mammalian cell culture systems to synthesize predetermined AIDS-associated polypeptides and derivatives and amino acid sequence variants thereof. Derivatives of AIDS-associated polypeptides include unglycosylated or variantly glycosylated polypeptides, as well as formylmethionyl N-terminal species.
Variants of normal AIDS-associated virus polypeptides are provided wherein one or more amino acid residues of the normal polypeptides have been deleted or substituted by other residues, or one or more amino acid residues inserted. These variants include predetermined fragments of normal polypeptides (deletion variants), fusion polypeptides containing an AIDS-associated virus polypeptide or fragment thereof with a second polypeptide which is not AIDS-associated virus polypeptide or fragment thereof (fusion or insertional variants), and all of the above in which an amino residue has been substituted. Preferred deletion variants are gp41 or gp160 envelope proteins from which one or more hydrophobic regions have been deleted.
DNA encoding the polypeptides of this invention are provided, as well as vectors operably incorporating such DNA and mammalian or prokaryotic cell cultures transformed therewith.
The predetermined polypeptide sequences of an AIDS-associated retrovirus produced herein are essentially free of other naturally occurring AIDS related polypeptides. These polypeptides contain at least one antigenic determinant which is capable of specifically binding complementary antibody. Specific polypeptide sequences described herein are designated p-24, p-15, Exe2x80x2, reverse transcriptase and envelope (env) polypeptides. In preferred embodiments, fragments and/or fusions of predetermined polypeptide sequences of an AIDS associated retrovirus, containing at least one antigenic determinant capable of specifically binding complementary antibody, are provided. In one species of this embodiment, a C-terminal portion of the p-24 and the entire p-15 sequence is removed. This truncated p-24, expressed in E. coli, demonstrates an unexpected reactivity with antibody to an AIDS associated retrovirus. Particularly preferred fragments are those of the env protein gp120 which are able to bind normal host cell receptors in competition with the intact virus, as well as Exe2x80x2 polypeptide fragments.
The fusion polypeptides comprise (a) a predetermined polypeptide sequence of an AIDS associated retrovirus or fragment thereof having at least one antigenic determinant capable of specifically binding complementary antibody and (b) a second polypeptide sequence which is not immunologically reactive with antibodies normally present in a biologically derived sample which is to be assayed for the presence of antibodies to AIDS associated retrovirus. If the fusion polypeptide is to be used as a component of a vaccine the second polypeptide sequence also should be incapable of inducing antibodies which are cross-reactive with polypeptides which are naturally occurring in the subject such vaccine is directed to. In this case, polypeptides from lower eukaryotes other than from yeast are useful.
In one species of a fusion polypeptide, DNA sequences encoding a polypeptide sequence of human growth hormone (HGH) are positioned five prime to a DNA sequence encoding the p-24 and p-15 polypeptide sequence of HTLV-III. This is expressed and processed in E. coli as an HGH-p24 fusion polypeptide which specifically binds complementary antibody. Similarly, fusions of Exe2x80x2 and env polypeptides such as gp41 and gp120 with viral, synthetic or prokaryotic polypeptides are provided. A preferred fusion is the fusion of a mature envelope polypeptide or fragment thereof with a signal sequence heterologous to the HTLV-III retrovirus, typically a eukaryotic or other viral polypeptide signal sequence. These results demonstrate that viral antigens associated with AIDS may be expressed as a fusion polypeptide to detect naturally occurring antibodies produced by AIDS infected individuals.
The immunoprotective polypeptide sequences of this invention are formulated into therapeutically effective dosages with a pharmaceutically acceptable vehicle and administered to AIDS-susceptible animals in order to induce the production of antibodies to such polypeptide sequences.
In a contemplated embodiment, a polypeptide comprising or consisting of a predetermined polypeptide sequence of RNA dependent DNA polymerase (reverse transcriptase) from an AIDS associated retrovirus is cloned and expressed for use in an assay system to identify compounds which inhibit such AIDS associated reverse transcriptase. Such compounds may be administered as a pharmaceutical agent to inhibit infection by AIDS associated retrovirus or dissemination of such retrovirus in infected individuals.